A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation

Abstract

The actin binding protein filamin A (FLNA) is required for somatostatin receptor 2 (SSTR2) and dopamine receptor 2 (DRD2) expression and signaling in GH- and PRL-secreting PitNETs, respectively, playing a role in tumor responsiveness to somatostatin receptors ligands and dopaminergic drugs. FLNA functions are regulated by several mechanisms, including phosphorylation. It has been shown that in GH-secreting PitNETs FLNA phosphorylation on Ser2152 (P-FLNA) switches FLNA function from a scaffold that allows SSTR2 signal transduction, to a signal termination protein that hampers SSTR2 antitumoral effects. Aims of the present study were to evaluate in PRL- and ACTH-secreting PitNETs cell lines MMQ and AtT-20 the effects of cAMP pathway activation and DRD2 agonist on P-FLNA and the impact of P-FLNA on DRD2 signal transduction. We found that forskolin increased (+2.2 ± 0.8-fold, p < 0.01 in MMQ; +1.9 ± 0.58-fold, p < 0.05 in AtT-20), and DRD2 agonist BIM53097 reduced (-49.4 ± 25%, p < 0.001 in MMQ; -45.8 ± 28%, p < 0.05 in AtT-20), P-FLNA on Ser2152. The overexpression of a phosphomimetic (S2152D) FLNA mutant in both cell lines prevented DRD2 antiproliferative effects, that were comparable in cells transfected with empty vector, wild-type FLNA as well as phosphodeficient FLNA mutant (S2152A) (-20.6 ± 5% cell proliferation, p < 0.001 in MMQ; -36.6 ± 12%, p < 0.01 in AtT-20). Accordingly, S2152D FLNA expression abolished the expected ability of BIM53097 to increase or decrease, in MMQ and in AtT20 respectively, ERK phosphorylation, an effect that was maintained in S2152A FLNA expressing cells (+1.8 ± 0.65-fold, p < 0.05 in MMQ; -55 ± 13%, p < 0.01 in AtT-20). In addition, the inhibitory effects of DRD2 on hormone secretion (-34.3 ± 6% PRL, p < 0.05 in MMQ; -42.8 ± 22% ACTH, p < 0.05 in AtT-20, in cells expressing S2152A FLNA) were completely lost in S2152D FLNA transfected cells. In conclusion, our data demonstrated that cAMP pathway and DRD2 agonist regulated FLNA activity by increasing or decreasing, respectively, its phosphorylation. Moreover, we found that P-FLNA prevented DRD2 signaling in PRL- and ACTH-secreting tumoral pituitary cell lines, suggesting that this FLNA modification might represent a new regulatory mechanism shared by different GPCRs. In PitNETs expressing DRD2, modulation of P-FLNA might suggest new pharmacological strategies to overcome drug resistance, and P-FLNA might represent a new biomarker for tumor responsiveness to dopaminergic agents.

Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant

“…Culture media were then collected to detect secreted ACTH. Murine ACTH levels were determined using a specific Elisa immunoassay kit (Fine Test, Wuhan Fine Biotech Co., Ltd., Wuhan, China), according to the manufacturer's instructions and previously reported protocols [26]. Absorbance was read at 450 nm in a Victor2 multilabel plate reader (Perkin Elmer, Whaltam, MA, USA).…”

Molecular mechanisms involved in somatostatin receptor regulation in corticotroph tumors: the role of cytoskeleton and USP8 mutations

“…The five subtypes of SSTRs differ in their patterns of β-arrestin mobilization and endosomal sorting. In particular, SSTR2 strongly and stably interacted with both β-arrestin 1 and 2, whereas SSTR5 transiently recruited only β-arrestin 2 (Tulipano et al 2004, Peverelli et al 2008, Treppiedi et al 2020). The interaction with β-arrestin 2 has a crucial role in the regulation of SSTR5 in GH-secreting PitNETs (Peverelli et al 2009, Peverelli et al 2013.…”

“…In addition, FLNA seems to play a role in SSTR5 downregulation induced by long-term pasireotide incubation in AtT-20 cells since in the absence of FLNA, pasireotide treatment did not affect the amount of SSTR5 (Treppiedi et al 2021a). On the contrary, in GH-secreting pituitary tumor cells, FLNA silencing did not affect SSTR2 levels, and FLNA is required to maintain SSTR2 stability after prolonged agonist stimulation by preventing lysosomal degradation and promoting efficient internalization and recycling to the plasma membrane of endocytosed SSTR2 (Peverelli et al 2014, Treppiedi et al 2020.…”

“…2:1 (Peverelli et al 2018, Mangili et al 2020. The impact of FLNA phosphorylation on SSTR5 expression and activity in tumoral corticotrophs, as well as the phosphorylation status of FLNA in ACTH-secreting PitNETs, remains to be determined.…”

Role of filamin A in the pathogenesis of neuroendocrine tumors and adrenal cancer

“…Indeed, FLNA alterations profoundly impacted on the SSTR2-mediated biological responses in both human GH-secreting PitNET primary cultured cells and GH3 cells. In the first case, FLNA knockdown abolished SSTR2-induced reduction of cyclin D1 and activation of caspase 3/7, required for reduction of cell proliferation and induction of cells apoptosis, respectively (Peverelli et al In GH-secreting pituitary tumor cells, the activity of FLNA is influenced by cAMP/PKA-mediated phosphorylation at amino acid residue Ser2152, as observed in PRL-and ACTH-secreting cells (Mangili et al 2021). FLNA phosphorylation was found to be upregulated or downregulated following forskolin or SSTR2 agonist incubation, respectively (Peverelli et al 2018b).…”